Inhibition of the ATP synthase sensitizes Staphylococcus aureus towards human antimicrobial peptides

Antimicrobial peptides (AMPs) are an important part of the human innate immune system for protection against bacterial infections, however the AMPs display varying degrees of activity against Staphylococcus aureus. Previously, we showed that inactivation of the ATP synthase sensitizes S. aureus towards the AMP antibiotic class of polymyxins. Here we wondered if the ATP synthase similarly is needed for tolerance towards various human AMPs, including human β-defensins (hBD1-4), LL-37 and histatin 5. Importantly, we find that the ATP synthase mutant (atpA) is more susceptible to killing by hBD4, hBD2, LL-37 and histatin 5 than wild type cells, while no changes in susceptibility was detected for hBD3 and hBD1. Administration of the ATP synthase inhibitor, resveratrol, sensitizes S. aureus towards hBD4-mediated killing. Neutrophils rely on AMPs and reactive oxygen molecules to eliminate bacteria and the atpA mutant is more susceptible to killing by neutrophils than the WT, even when the oxidative burst is inhibited.These results show that the staphylococcal ATP synthase enhance tolerance of S. aureus towards some human AMPs and this indicates that inhibition of the ATP synthase may be explored as a new therapeutic strategy that sensitizes S. aureus to naturally occurring AMPs of the innate immune system.

www.nature.com/scientificreports/ oral health by limiting infections in the oral cavity 5 . Several histatins have been characterized, however histatin 5 displays the strongest antimicrobial activity 5 . LL-37 is an antimicrobial peptide that belongs to the cathelicidin family and is expressed in various epithelial-(e.g. keratinocytes) and immune cells (e.g. neutrophils and macrophages) 7 . The bactericidal activities of many AMPs have generally been attributed to pore formation in bacterial cytoplasmic membranes, however this mode of action may be too simplistic 8,9 . The bactericidal activity of hBD3 has for example been associated with lipid II binding, leading to inhibition of cell wall biosynthesis 10 and some AMPs also have intracellular targets 11 . Many antimicrobial peptides display a net positive charge, which is important in the initial electrostatic attraction to negatively charged bacterial phospholipid membranes and negatively charged teichoic acids on the surface of Gram positive bacteria, e.g. Staphylococcus aureus 11 . S. aureus is a common colonizer of the human body 6 , where approximately 30-50% of healthy adults transiently carry this species and approximately 20% are persistently colonized 12 . The skin, nose and intestinal tract are important ecological niches for S. aureus carriage 13 . Topical colonization with S. aureus imposes a risk for subsequent infections, if the skin or mucosal barriers are breached and enables transmission of S. aureus cells to the adjacent tissues or the bloodstream 12 . S. aureus is an opportunistic pathogen that may cause life-threatening diseases, such as sepsis, endocarditis and pneumonia 12 . Even though keratinocytes express various antimicrobial peptides, such as hBD1-4 and LL-37, S. aureus frequently colonizes human skin 6 . Among the human β-defensins, only hBD3 displays potent bactericidal activity against S. aureus at physiological conditions [14][15][16] , and keratinocytes are dependent on hBD3 for killing of S. aureus 17 . However, it is incompletely understood, why the remaining β-defensins display limited anti-staphylococcal activity. This indicates that sensitizing S. aureus towards the innate immune system AMPs may potentially facilitate eradication of colonizing S. aureus.
Multiple factors affect bacterial susceptibility towards AMPs, such as cell membrane composition, cell surface charge and transmembrane potential 8 . The positive charge of many AMPs facilitates the interaction with negatively charged bacterial envelopes 11 . A common resistance mechanism exploited by bacteria is to reduce the net negative charge of the cell envelope, for example by lysinylation of phospholipids 18 and D-alanylation of teichoic acids in S. aureus 19 . Curiously, deficiency of wall teichoic acids selectively confers reduced susceptibility to hBD3, while not affecting susceptibility to LL-37 and HNP1-3 20 . The transmembrane potential affects the ability of cationic AMPs to permeabilize membranes 8,21 , where an inside-negative transmembrane potential facilitates insertion of some cationic AMPs into bacterial membranes 8 . Interference with the electron transport chain by inactivation of menaquinone-(men mutants) or hemin (hem mutants) biosynthesis pathways leads to membrane depolarization in S. aureus and in the appearance as small colony variants (SCVs) on agar plates 22 . Electron transport chain SCVs have been associated with reduced susceptibility towards multiple AMPs, including hBD2-3 23 , thrombin-induced platelet microbicidal protein 24 and nisin 24 . We recently reported that inactivation of genes encoding for multiple subunits of the ATP synthase sensitizes S. aureus towards polymyxins 25 , a class of cationic AMPs that is used for treatment of Gram-negative infections 26 . The ATP synthase basically serves two physiological functions, first being synthesis of ATP from ADP and inorganic phosphate by using energy from the proton motive force. Secondly, during conditions with a low proton-motive force the ATP synthase can work in reverse as an ATPase and thereby contributes to the establishment of a cross-membrane proton gradient through ATP hydrolysis 27 . In S. aureus, the ATP synthase is primarily used to hydrolyze ATP for maintaining the cross-membrane proton gradient both under fermentative and respiratory conditions 28 . ATP synthase inactivation in S. aureus leads to hyper-polarization of the membrane 25,28 , which was hypothesized to be the mechanism for sensitizing ATP synthase mutants towards polymyxins 25 .
Several molecules have been shown to inhibit the ATP synthase in different species 29 . For example inhibition of the ATP synthase with oligomycin A sensitizes S. aureus towards polymyxin B 25 and aminoglycosides 30 . However, oligomycin A displays similar 50% inhibitory concentration (IC 50 ) between S. aureus and human mitochondrial ATP synthases 31 , and cannot be used clinically due to toxicity issues 32 . Resveratrol is a widely used nutraceutical that has been shown to bind to the bovine ATP synthase in the F 1 -domain 33 and also binds reversibly to the ATP synthase in E. coli, partially inhibiting both ATP hydrolysis and ATP synthesis 34 . Coadministration of resveratrol sensitizes S. aureus towards aminoglycosides 30 . However, resveratrol is readily metabolized following oral administration, which probably only enables topical use 35 .
Since ATP synthase inactivation sensitizes S. aureus towards polymyxins, we hypothesize that this strategy also can sensitize S. aureus towards various human AMPs. Potentiation of human AMPs that are currently ineffective against S. aureus may potentially become a new therapeutic strategy, where treatment relies on deprivation of AMP resistance mechanisms and hence boosting of the naturally occurring AMPs of the innate immune system.

Materials and Methods
Bacterial strains, growth conditions and chemicals. The Staphylococcus aureus JE2 wild type (WT) strain and derivative mutants used in this study are highlighted in Table 1. Antimicrobial peptides used in this study included histatin-5 (Innovagen, Sweden), LL-37 (Isca Biochemicals, United Kingdom) and hBD1-4 (Innovagen, Sweden), as well as polymyxin B Etests (bioMérieux, France). We used the ATP synthase inhibitor resveratrol (Santa Cruz Biotechnology). Bacterial strains were routinely cultured at 37 °C in tryptic soy broth (TSB) or on tryptic soy agar (TSA).
Antimicrobial susceptibility assays. Microdilution. The minimum inhibitory concentration for resveratrol was determined using a two-fold broth microdilution assay in TSB (100 µl) with an initial inoculum of approximately 5 × 10 5 cells/ml. MIC was determined upon incubation at 37 °C for 24 h.
Etest. The MIC for polymyxin B was determined using Etest (bioMérieux, France) in the absence and in the presence of sub-inhibitory concentrations (0.0625x-0.25 × MIC) of resveratrol or menadione (1 µg/ml, Sigma). Bacterial cell survival assays. From overnight cultures of S. aureus JE2 and mutants, 10 μl was diluted into 990 μl fresh TSB medium in a falcon tube and grown for 2 h for the cells to reach early exponential phase. After 2 h the cultures were re-suspended in 10 mM sodium phosphate buffer (pH 7.4), termed NaPi (Medicago, Sweden). Cells were subsequently diluted in NaPi to approximately 5 × 10 5 CFU/ml, and combined with antimicrobial peptides and resveratrol when indicated, to a final volume of 100 µl and incubated in 96-well plates with shaking for 2 h at 37 °C. Bacteria were plated for CFU on TSA plates. Following overnight incubation at 37 °C for 24 h, viable cells were enumerated and relative cell survival was calculated as CFU with peptide /CFU without peptide at 2 h post-exposure. Values provided are the mean ± SEM derived from at least three independent biological replicates.
isolation of pMns from human blood. Blood was collected from healthy adult volunteers and written informed consent was given. Isolation of neutrophils was performed following the procedure described in 36 . All methods were carried out in accordance with relevant guidelines and regulations. The institutional review board (IRB) of the University of Tübingen approved the study and all adult subjects provided informed consent. This study was done in accordance with the ethics committee of the medical faculty of the University of Tübingen that approved the study, Approval number 015/2014 BO2. Briefly, heparinized blood was diluted 1:1 (v/v) with PBS containing 0.5% BSA and 2 mM EDTA and layered onto a gradient of Biocoll (density, 1.077 g/ml; Biochrom) and Histopaque (density, 1.119 g/ml; Sigma). After centrifugation for 20 min at 380×g, neutrophils were collected from the Histopaque phase. Cells were subjected to a brief hypotonic shock with ultrapure-water containing 155 mM ammonium chloride, 1 mM potassium hydrogen carbonate and 0. After incubation, the plate was centrifuged at 300×g for 10 min, the supernatants were collected, and the remaining neutrophil pellets were lysed using cold ddH 2 0 for 10 min on a rocker. The lysed neutrophils and remaining bacteria were resuspended and pooled with the previously collected supernatants. Dilutions of 10 -2 were plated on TSA plates with an Eddy Jet 2 W and incubated overnight at 37 °C. Values provided are the mean ± SEM derived from at least seven independent biological replicates.

Results
ATP synthase mutants are more susceptible to hBD2 and hBD4. Since inactivation of the ATP synthase increases susceptibility of S. aureus towards the antimicrobial peptides, polymyxins 25 , we wondered if inactivation of the ATP synthase also sensitizes S. aureus towards AMPs of the human innate immune system. Therefore, bacterial killing of the WT S. aureus JE2 and isogenic atpA (ATP synthase subunit alpha) transposon mutant was assessed following 2 h exposure to human cationic AMPs, comprising histatin-5, LL-37 and hBD1-4 at the concentrations highlighted in Fig. 1a. The atpA mutant was more susceptible to hBD4 and hBD2 compared with the WT (Fig. 1a). The atpA mutant displayed a 63-fold greater reduction in viable cells compared with WT upon treatment with hBD4. For hBD2, the atpA mutant displayed a fivefold greater reduction in viable cells compared with WT. A minor increase in bactericidal activity against the atpA mutant was detected for histatin-5 and LL-37, whereas no differences between WT and atpA survival were detected upon treatment with hBD1 nor hBD3.
We assessed hBD4-mediated killing of other ATP synthase mutants, namely atpB (ATP synthase subunit A) and atpG (ATP synthase subunit gamma) and both mutants displayed increased susceptibility to hBD4, similarly to the atpA mutant (Fig. 1b).
Since the atpA mutant has a hyperpolarized membrane 25 , we also assessed hBD4 susceptibility of the menD transposon mutant, which has a depolarized membrane 37 . The menD mutant was indeed more tolerant to hBD4 compared with WT, as no reduction in viable cell count was observed following 2 h exposure to hBD4 at 5 µM (Fig. 1b), suggesting that the magnitude of the membrane potential is an important determinant for hBD4 susceptibility.  Table S1). The menD mutant is auxotrophic for menadione and supplementation with the compound re-sensitized the menD mutant to polymyxin B (Supplementary Table S1).
Taken together, inactivation of the ATP synthase sensitizes S. aureus to specific human AMPs and the magnitude of the membrane potential correlates with hBD4 susceptibility. This correlation also applies more broadly to include the non-human AMP, polymyxin B.
the Atp synthase inhibitor resveratrol sensitizes S. aureus towards hBD4. Resveratrol is a putative ATP synthase inhibitor in S. aureus 30 and therefore, we assessed if resveratrol could sensitize S. aureus JE2 towards hBD4. Resveratrol has growth-inhibitory properties with a MIC of 256 µg/ml, but at a sub-inhibitory concentration (0.125 × MIC) it had no impact on S. aureus viability (Fig. 2). Importantly, however, when supplemented in combination with hBD4, resveratrol increased killing of WT S. aureus by 20-fold compared with hBD4 alone.
These result suggests that ATP synthase inhibition with resveratrol may be an attractive approach to sensitize S. aureus towards hBD4.
the atpA mutant is more susceptible to killing by human neutrophils. Log-phase bacteria of the WT and atpA mutant were opsonized with pooled normal human serum, phagocytosed by neutrophils, and subsequently incubated for one hour before determination of surviving cells. The atpA mutant was more susceptible to neutrophil killing than the WT and after one hour of incubation with neutrophils, only 39.2% of the atpA cells survived compared with 49.9% for the WT (P = 0.006) (Fig. 3). The uptake of the strains into the neutrophils was similar (Data not shown), suggesting that the increased killing of the atpA is due to antimicrobial activities of the neutrophils and not due to alterations in phagocytosis rates.
As neutrophils normally use both, oxygen-dependent and non-oxygen-dependent killing mechanisms, including antimicrobial peptides 38 , we compared survival of atpA and WT in neutrophils treated with the NADPH oxidase inhibitor diphenyleneiodonium (DPI), which suppresses the formation of reactive oxygen species 18 . Suppression of the oxidative burst limited killing of S. aureus (Fig. 3). After one hour incubation only 74.3% of the atpA cells survived in DPI-treated neutrophils, compared to 91.4% for the WT (P = 0.0196) (Fig. 3). These data suggest that the atpA mutant is more susceptible towards the oxygen-independent antimicrobial activities of neutrophils.

Discussion
Antimicrobial peptides are an important part of the innate immune system and the AMPs display activity against a wide range of bacterial-, fungal-and viral species 6 . Several human AMPs however display low inhibitory activity against S. aureus 14 . For example, the human β-defensins 1-4 are produced by keratinocytes and are key in protecting against skin infections 6 . hBD3 displays greater bactericidal activity against S. aureus than the other β-defensins [14][15][16] , and hBD3 is important for keratinocytes in killing S. aureus 17 . Production of AMPs in the skin and in the nasal passages plays a major role in preventing S. aureus persistent colonization and people with defects in hBD3 production have enhanced nasal colonization of S. aureus 39 . Our results point to a novel www.nature.com/scientificreports/ type of antimicrobial therapy, whereby the susceptibility of the pathogen is enhanced towards the natural human antimicrobial peptides. Here we demonstrate the potential for S. aureus, but it may be applicable to other human pathogens as well.
The energetic state of bacterial membranes can affect the susceptibility towards AMPs in different bacterial species 8 and for some conventional classes of antibiotics as well, i.e. aminoglycosides 40 . Recently, we demonstrated that ATP synthase mutants of S. aureus become more sensitive towards polymyxins 25 . ATP synthase inactivation confers hyperpolarization of the membrane 25,28 and larger membrane potentials can facilitate AMP insertion into membranes 8 . In this study, we demonstrate that the activities of certain human AMPs are affected by the magnitude of the membrane potential. ATP synthase mutants have a hyperpolarized membrane 25,28 and become more sensitive towards hBD2 and hBD4 and to a minor degree towards LL-37 and histatin-5 (Fig. 1a). Contrarily, a menD mutant with a depolarized membrane is less sensitive towards hBD4 (Fig. 1b).
Our finding that membrane depolarization protects S. aureus from hBD4-mediated killing corroborate previous studies demonstrating that S. aureus SCVs are less susceptible to different AMPs. For S. aureus, electrontransport chain deficient mutants are less susceptible to killing by thrombin-induced PMP-1 (tPMP-1) 41 , nisin 24 , lactoferrin B 42 and human AMPs, including hBD2, hBD3 and LL-37 23 . Another study, with genetically defined menD and hemB mutants in different S. aureus genetic backgrounds did however not observe changes in susceptibility to LL-37 43 . Killing by the human neutrophil defensin 1 (hNP-1) is similar in WT and mutants with impaired electron transport chains 24,41,44 . These observations indicate that membrane potential changes only affect the activity of specific AMPs.
It has been suggested that membrane depolarization and subsequently increased tolerance towards AMPs of the innate immune system is a survival strategy that enable intracellular persistence of S. aureus in eukaryotic cells 45 . Here we demonstrate that inactivation of the ATP synthase contrarily sensitizes S. aureus to neutrophilkilling (Fig. 3). The increased susceptibility to neutrophil-killing is also evident when the oxidative burst is suppressed (Fig. 3), suggesting that this effect is mediated by increased susceptibility to AMPs produced by neutrophils 38 .
It is not only in S. aureus that AMP sensitivity is modulated by the magnitude of the membrane potential. In E. coli, inactivation of the ATP synthase also leads to hyperpolarization of the membrane 46 and ATP synthase mutants are more sensitive to colistin 47 and aminoglycosides 40,47 . Deletion of the gene phoP in E. coli conferred hyperpolarization of the membrane and a concomitant increase in activity of polymyxin B, while collapsing the proton gradient with the protonophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP) abrogated this effect 48 . Dissipation of membrane potential with CCCP also impaired killing of E. coli with the AMP indolicidin 49 . For Salmonella enterica Typhimurium, impairment of the electron transport chain reduces AMP activity, e.g. a hemB mutant displays a fourfold increase in MIC for colistin 50 . Even respiration-deficient mutants of the fungus Candida albicans experience reduced sensitivity to histatin-5 and chemical inhibition of the electron transport chain with sodium azide or CCCP treatment also protects C. albicans against histatin-5 killing 51,52 . www.nature.com/scientificreports/ The ATP synthase may potentially be targeted to facilitate killing by AMPs of the innate immune system and hence be essential under in vivo conditions. By employing the Tn-seq methodology, the ATP synthase has been identified in several studies as essential during in vivo conditions, such as in abscess formation or osteomyelitis, while the ATP synthase is dispensable during growth in laboratory medium [53][54][55] . Recently, Grosser and colleagues demonstrated that an ATP synthase mutant indeed is severely attenuated in a murine skin abscess model 28 . ATP synthase inactivation confers pleiotropic effects, including attenuated growth under anaerobic conditions, increased sensitivity towards peroxide and nitric oxide stresses 28 . Whether virulence attenuation of the ATP synthase mutant in the murine skin abscess model is mediated by a single phenotypic trait or a combination thereof remain unexplored in the study 28 and here we provide an additional phenotype that may contribute to the attenuated virulence, namely increased sensitivity of S. aureus to different AMPs of the innate immune system.
Inhibition of the ATP synthase may potentially have therapeutic value either as a monotherapy or in combination with AMPs or aminoglycosides. Many ATP synthase inhibitors have been identified 29 , however several of these, e.g. oligomycin A, are not selective for bacterial ATP synthases and also blocks human mitochondrial ATP synthases 31 . The ATP synthase has been clinically validated as a druggable target in recent years with the antibiotic bedaquiline that selectively inhibits ATP synthases of Mycobacteria 56 . We demonstrated that the ATP synthase inhibitor resveratrol, a commonly used nutraceutical 57 , sensitizes S. aureus towards hBD4 (Fig. 2). Resveratrol has previously been assessed for clearance of acne skin lesions 58 and has recently been shown to reduce abscess formation by S. aureus when used as a monotherapy 59 . It will be important in future animal experiments to elucidate, whether resveratrol in combination with human defensins are superior in treating topical S. aureus infections compared with the respective monotherapies.
It is encouraging that bacterial ATP synthases are sufficiently different from human ATP synthases, which enables identification of selective bacterial ATP synthase inhibitors that are not toxic to human cells 32 . Selective staphylococcal ATP synthase inhibitors may provide a novel class of antibacterial therapies that is based on sensitizing S. aureus towards the AMPs of the innate immune system. Additionally, such inhibitors can be adjuvants that potentiate the activity of conventional antibiotics, such as aminoglycosides and polymyxins 25,30 . Several AMPs are in clinical development 3 , whose activity potentially also can be enhanced by co-administration of ATP synthase inhibitors.
In summary, we have investigated the possibility of sensitizing S. aureus towards human AMPs by targeting the ATP synthase and our results suggest that it may be a novel strategy for development of new antimicrobial therapeutics.